Efficient Characterization for Protein Crystals Using Confocal Raman Spectroscopy

Abstract

Confocal Raman spectroscopy was applied to the characterization of various states emerging in the screening of protein crystallization. Four main characterized states, namely single crystals, microcrystals, precipitates, and clear drops without solid materials, appear in a droplet for crystallization; the first three states should be critically distinguished and characterized because of the limitations of visual observation under an optical microscope. Using lysozyme and other proteins, crystallization was performed by the hanging drop vapor diffusion technique and was monitored through an automated confocal Raman system. Prior to the spectroscopic analysis, an optical microscope with a charge-coupled device (CCD) camera and associated image processing software were used to rapidly identify the XY locations to be measured spectroscopically by focusing the laser beam on a test sample. Instead of the current image analysis by optical microscopy, confocal Raman spectroscopy with a high spatial resolution was used to identify the state of protein crystallization. Such real-time Raman monitoring also distinguished real protein crystals from pseudo-protein crystals emerging in a crystallization droplet.

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“…Finally, we investigated the local change or inhomogeneity of the concentrations inside a protein drop for both the setups of vapour diffusion and evaporation stimulation with the assistance of the high spatial resolution of the confocal system (i.e., 0.76/5.0 µm for lateral/axial resolutions, respectively) 8 . Confocal Raman spectra were acquired at various locations inside a protein drop using only a 10 s exposure time to ensure that the collection time was short enough to neglect the influence of crystallization process from changes in the observed spectral features.…”

“…In this respect, Raman monitoring has demonstrated a considerable potential for in-situ investigation of the compositional changes in a hanging drop throughout the crystallization process, which leads to insight into the degree of supersaturation and the crystallization www.scienceasia.org mechanism. [5][6][7] It is also possible to use a confocal Raman microscope combined with an XY piezo-stage modified specifically to observe crystallization plates 8 . Such a combination permits the simultaneous measurement of both spectroscopic information and a microscopic image from a small local area within a drop.…”

“…The confocality provides an extremely small sampling volume in the submicron range with a significant reduction in background signals coming from areas encircling the laser focus point. As a consequence, a confocal Raman spectrum of a protein crystal or even a microcrystal with a diameter less than 10 µm can be obtained at a high spectral signal-to-noise ratio within a minute 8 . In addition to the monitoring techniques, the development of the crystallization procedure itself is important to provide effective crystallization.…”

Raman characterization and chemical imaging of biocolloidal self-assemblies, drug delivery systems, and pulmonary inhalation aerosols: A review

“…Confocal Raman microspectroscopy 55 has been reported to have great potential for efficiency in assessing and quantifying the physical state of a drug, physicochemical stability of the formulation, content, and homogeneity of drug distribution in a polymer formulation matrix. 56 Additionally, confocal Raman microspectroscopy has been employed in protein crystallization, 57 molecular imaging of the effects of penetration enhancers and drugs on in vivo human stratum corneum, [58][59][60] and in vivo human skin analysis. 61 With an elegant combination of confocal Raman spectroscopy with laser tweezers (ie, optical trapping to isolate single cells in a more natural state, as opposed to adsorption onto the surface of a solid substrate, which is known to disturb the natural state), rapid identification of single live cells (bacterial cells and fungal spores) and organelles can be accomplished from every angle.…”

Micro ATR FTIR imaging of hanging drop protein crystallisation

“…As such a drop of protein solution and precipitant solution can be deposited directly onto the Ge crystal and a reservoir can then be placed underneath the drop in exactly the same manner as that which is applied in structural biology labs. Confocal Raman microscopy has also been applied to the in situ measurement of hanging drop protein crystallisation to monitor the change in concentration of protein within the crystallisation drop [24,25]. Micro ATR FTIR imaging is an imaging technique and thus requires shorter measurement and sample preparation times to obtain the same number of spectra from the sample in comparison with Confocal Raman microscopy.…”